Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease of 2019 (COVID-19) pandemic, which has infected millions of people worldwide. To date, several vaccines and antivirals have been developed against SARS-CoV-2; however, its tendency to mutate rapidly poses a continued threat to human health. As such, the development of better pan-coronavirus therapeutics is still required. Recently, the SARS-CoV-2 non-structural protein 13 (Nsp13) helicase has been shown to be an attractive therapeutic target given its high conservation rate among coronaviruses and indispensable role in viral replication. Based on this, we sought to further study the biochemical mechanisms behind Nsp13's binding and unwinding activities, along with its interactions with host cells, to provide further insight for future therapeutic development. To study the binding and unwinding activity of Nsp13, we site-specifically incorporated the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into Nsp13 to act as a bioorthogonal handle for fluorescent labelling. We identified five potential sites for AzF incorporation in Nsp13 and assessed their reactivities towards a conjugated Cy5 fluorophore through strain-promoted alkyne-azide cycloaddition (SPAAC). Further experiments were also performed to ensure that the unwinding activity was not adversely affected before these Nsp13-AzF constructs were utilized in fluorescence resonance energy transfer (FRET)-based binding assays. Ultimately, the F81AzF construct was identified to be the most suitable for monitoring the binding of Nsp13 to a series of nucleic acid substrates in a distance-dependent manner by FRET. The next steps of this project would be to implement F81AzF in single-molecule FRET (smFRET) experiments to directly monitor the positioning and dynamics of this helicase on its substrate. In addition, interactions between Nsp13 and host cellular and biological processes were investigated to provide further insight into potential mechanisms that can be exploited for novel therapeutic development. From transcriptomic profiling analyses of A549 cells, we uncovered that Nsp13 influences microRNA (miRNA) expression and signalling pathways; in particular, miR-146a, a potent mediator of inflammation and immune responses, was found to be induced upon Nsp13-overexpression. Further experiments revealed that this may lead to the inhibition of NF-κB signalling, through the repression of the upstream targets TRAF6 and IRAK1, to suppress the production of proinflammatory cytokines and facilitate viral infection. Collectively, from this work, we propose that further exploration of these miR-146a-mediated signalling pathways may present alternative strategies for antiviral investigations.